We have shown that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery (i.e., transposon and transposase are on separate plasmids) is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilization, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed(+) T cell clones were generated and found to be negative for the transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was positive for transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007-0.047 per cell) by quantitative PCR. Transposase-positive bulk T cells lacked transposase plasmid demonstrated by Hirt (episomal) extracted DNA and showed no detectable transposase by Southern hybridization, Western blot, and quantitative RT-PCR analyses. Cytogenetic and array comparative genomic hybridization analyses of the only identified transposase-positive clone (O56; 0.867 copies per cell) showed no chromosomal abnormality or tumor formation in nude mice although transposon remobilization was detected. Our data suggest that SB delivery via plasmid in T cells should be carried out with caution because of unexpectedly high copy numbers of randomly integrated SB transposase.